SUMMARY With a lack of druggable oncogenic mutations in small cell lung cancer (SCLC) and no approved targeted therapies, we need to identify new treatment approaches for SCLC. In this proposal, we apply whole genome CRISPR inactivation screens to identify genes that are essential for SCLC broadly, or for key genetically defined subsets. We then determine whether pharmacologic inhibition of identified hits also results in selective killing of SCLC cells. We apply in vivo systems including patient derived xenograft (PDX) and genetically engineered mouse models to test pharmacologic inhibitors of key targets identified in these genetic screens. Aim 1 will focus on a pathway that we already identified using CRISPR inactivation screens to be essential for Rb/p53-mutant SCLC cells. Components of a NEDD8/RBX1 pathway, controlling Cullin activity and protein ubiquitylation, were essential for SCLC cells. Pharmacologic inhibition of NEDD8 activating enzyme (NAE) using MLN4924 in patient derived xenograft (PDX) models confirmed reliance of SCLC cells on this pathway. Excitingly, we identified examples of exceptional responses to MLN4924 in some PDX models. Aim 1: Apply in vivo models to direct NEDD8 inhibition to subsets of SCLC most likely to respond. Here, we explore the potential for inhibiting the NEDD8 pathway in SCLC. We will combine NAE inhibition with cisplatin-etoposide chemotherapy and determine whether durable responses result. Some SCLC PDX models exhibited exquisite sensitivity to NAE inhibition; we will identify underlying mechanisms and genetic features of super-responding PDX models that could be used to help predict exceptional responders. We will determine whether NAE inhibition should be directed to all SCLC patients, or to key subsets, and whether this approach augments response to chemotherapy. MLN4924 is already being tested in clinical trials for other cancer types and this work could rapidly lead to clinical trials in SCLC. Aim 2: Apply CRISPR inactivation screens to identify new therapeutic targets for SCLC. This Aim will extend our CRISPR inactivation screens to include mouse SCLC cell lines with key driver mutations beyond Rb/p53, including Pten, Crebbp and Mycl. Our approach takes advantage of the fact that while human SCLC has an extremely high mutational burden, leading to extensive heterogeneity among SCLC with a given driver mutation, mouse SCLC with defined driver alterations are more homogenous, increasing signal to noise in identifying vulnerabilities. We will identify dependencies in genetically-defined subsets of SCLC and will test inhibitors of identified druggable targets in PDX and GEM models that harbor defined genetic alterations. We leverage available mouse models and derived cell lines, along with a bank of genetically annotated PDX models, studied in vivo and ex vivo, to identify and evaluate completely new ways to target key genetic subsets of SCLC.